The present invention relates to polypeptides that are useful for diagnosing American trypanosomiasis, or Chagas disease, a disease caused by the infectious agent Trypanosoma cruzi. More particularly, the invention relates to recombinant T. cruzi polypeptides, synthesized using genetic engineering techniques, and to constructs and processes for producing the recombinant polypeptides, and to an assay for detecting T. cruzi infection which employs the polypeptides.
American trypanosomiasis, or Chagas disease, is an illness caused by the protozoan parasite, T. cruzi (1,2). This organism is transmitted by insects called reduviid bugs (3), by blood transfusion (4), and also from mother to fetus (5). Several years after acquiring T. cruzi infection, patients may develop the cardiac and gastrointestinal symptoms that are associated with chronic infection, which is life-long, but the majority of infected persons never develop clinical manifestations of Chagas disease and are unaware of being infected. The two drugs available for treating T. cruzi infections have low efficacy and often cause serious side effects. In practice, therefore, they have virtually no impact on the control of Chagas disease.
Chagas disease is a major cause of morbidity and death in Latin America, where an estimated 16-18 million people are chronically infected with T. cruzi (6). In recent years tens of thousands of T. cruzi-infected people have emigrated to the United States, especially from Central America, where the prevalence of T. cruzi infection is high, thus creating the risk of transfusion-associated transmission of the parasite here (7-9). Several such cases have been described (10-12).
Since clinical criteria cannot be depended upon for recognizing T. cruzi infection, blood tests are of paramount importance, both in patient care settings and in blood banks. Chronically infected persons uniformly have anti-T. cruzi antibodies. The diagnosis of T. cruzi infection is almost always made by detecting these antibodies in patients' blood, since parasitological approaches are laborious and lack sensitivity and, as noted, clinical evaluations lack specificity.
Immunological tests currently used to diagnose T. cruzi infection, such as complement fixation and indirect immunofluorescence tests, and enzyme-linked immunosorbent assays (ELISA), often produce inconsistent results and false-positive reactions (13). The occurrence of false-positive reactions can be a problem with specimens from patients with leishmaniasis, schistosomiasis, and other parasitic and infectious diseases, with samples from patients with autoimmune disorders and other illnesses, and with specimens from normal persons.
In large measure these problems with sensitivity and specificity occur because the assays are based on antigens extracted from parasites grown in the laboratory. The complexity and variability of mixtures of native antigens derived from cultured parasites, which persist even after fractionation and purification procedures are used, have been a major barrier to standardization of immunoassays. Because of the limitations of these immunoassays, experts generally agree that blood samples should be positive in three different assays, performed in parallel, before being accepted as positive.
An additional problem related to assays based on material derived from cultured parasites is that producing the antigens creates a serious biohazard for technical personnel, and laboratory-acquired cases of Chagas disease occur with disquieting frequency, both in the United States and abroad (14,15). Furthermore, some of the immunoassays currently available require sophisticated laboratory equipment and levels of technical expertise not generally available in the countries in which T. cruzi infection is endemic.
In response to the need for improved assays for detecting T. cruzi infection, considerable work has been invested in the development of new immunoassays. These efforts have accelerated in recent years as new technologies have become available that have the potential for serving as the basis of improved assays. Recombinant DNA technology has led to the molecular cloning of several antigenic T. cruzi proteins. Cloned segments of T. cruzi genes have been used to produce in bacteria portions of antigenic proteins (16-22). In research settings several of these, singly and in combination, have been used as target antigens in immunoassays. These assays have not been tested in field or blood bank trials, and none is available commercially.
U.S. Pat. No. 4,870,006 discloses the use of a recombinant protein in an assay for diagnosing T. cruzi infection. A 70-kilodalton heat shock protein constitutes the target antigen in this assay. No information regarding the sensitivity and specificity of the assay is provided in the patent.
In this context, therefore, a need exists for a highly sensitive and specific system for detecting T. cruzi infection that is safe, easy, and inexpensive to manufacture and perform.